Serum, plasma, and dried blood spot high-sensitivity C-reactive protein enzyme immunoassay for population research

Abstract

C-reactive protein (CRP) is used as a biomarker of morbidity and mortality risk in studies of population health, and is essential to interpretation of several micronutrient biomarkers. There is thus a need for a robust high-sensitivity CRP (hsCRP) measurement method for large-scale, non-clinical studies. We developed an efficient, inexpensive assay suitable for quantifying CRP across the physiological range using any blood specimen type. The ELISA uses readily available monoclonal antibodies to measure CRP in serum, plasma, or dried blood spots (DBS) made from venous or capillary blood. Assay performance was evaluated by standard methods, including comparison with a previously described assay. Effects of specimen type were tested by measuring CRP in 52 matched serum, plasma, and venous and capillary dried blood spot specimens. Long- and short-term CRP stability were evaluated. Assessments of assay limits of detection, linearity, recovery, imprecision, and concordance with an established method (Pearson correlation=0.988, n=20) demonstrated the validity of the new assay. CRP measurements in serum, plasma, and DBS had Pearson correlations from 0.974 to 0.995, n=52, but CRP in serum was on average 1.6 times (SD 0.37) higher than in DBS. CRP was stable in frozen serum for up to 34 months, but DBS CRP declined quickly with exposure to ambient temperatures, and across long-term storage at -20°C. This hsCRP assay is a robust and inexpensive tool designed for use in large-scale population health research. Our results indicate that DBS CRP is less stable than previously reported.

Figure 1

Assay linearity for plasma (panel A) and venous DBS (panel B); n = 10 specimens per sample type.

Figure 2

Log CRP in serum, plasma, capillary DBS, and venous DBS specimens collected at the same time from 52 individuals. Broken line = line of equality. Solid line = linear regression. Unlogged minimum and maximum values across all sample types = 0.03 to 22.4 mg/L CRP.

Figure 3

Bland-Altman plots of differences between log serum CRP and log CRP in plasma (panel A), capillary DBS (panel B), and venous DBS (panel C) plotted against average log CRP concentration; n = 52 specimens per sample type. Center line indicates mean difference; upper and lower lines indicate the 95% confidence interval. Broken line indicates the line of equality.

Figure 4

Effect of long-term storage at −20°C on CRP in serum (panel A) and DBS (panel B); n = 8 specimens per sample type per time point. Values are CRP ± SE predicted by linear mixed effects models, with time as a factor. Decline over time was significant for DBS (p<.0001) but not for serum (p=.4).

Figure 5

Difference between log serum CRP and log CRP in capillary DBS plotted against days of DBS storage at ambient field temperatures in 23 matched sets of specimens collected and stored in a remote field setting. Center solid lines indicates mean difference for specimens stored less than and greater than 21 d at ambient temperatures, broken upper and lower lines indicate the 95% confidence intervals.